Tumor Suppressor miR-613 Alleviates Non-Small Cell Lung Cancer Cell via Repressing M2 Macrophage Polarization

Background Non-small cell lung cancer (NSCLC) is a crucial crux of cancer-related death, and M2 macrophage polarization facilitates NSCLC development. MicroRNA-613 (miR-613) is a tumor suppressor. This research aimed to clarify the miR-613 function in NSCLC and its impact on M2 macrophage polarization. Methods. miR-613 expressions in NSCLC tissues and cells were evaluated using quantitative real-time PCR. For miR-613 function in NSCLC, cell proliferation analysis, cell counting kit-8, flow cytometry, western blot, transwell, and wound-healing were conducted. Meanwhile, the miR-613 impact on M2 macrophage polarization was assessed by the NSCLC models. Results. miR-613 was lessened in NSCLC cells and tissues. It was corroborated that miR-613 overexpression retrained NSCLC cell proliferation, invasion, and migration but facilitated cell apoptosis. Moreover, miR-613 overexpression restrained NSCLC development by repressing M2 macrophage polarization. Conclusion Tumor suppressor miR-613 ameliorated NSCLC by restraining M2 macrophage polarization.


Introduction
Lung cancer is a malignant tumor worldwide with high frequency in morbidity and mortality [1]. Nearly, 85% of lung cancer is non-small-cell lung cancer (NSCLC) [2,3]. Although multiple progress has been achieved in NSCLC treatment, the efect is unsatisfactory [4,5]. Tus, there is an urgent need to better understand NSCLC pathogenesis and identify novel therapeutic targets to alleviate NSCLC.
In search for more efective intervention methods for NSCLC, the importance of targeting the tumor microenvironment (TME) has gradually attracted wide attention [6,7]. TME forms of cancer and stromal cells containing macrophages, and endothelial cells [8,9]. As one of the pivotal components of TME, tumor-associated macrophages (TAMs) are major coordinators of immune cells [10]. TAMs mainly include M1 and M2, and M2 macrophages accelerate tumorigenesis [11,12]. Crucially, accumulated studies demonstrate that M2 macrophages drive proliferation, migration, invasion, and other malignant phenotypes of NSCLC, thereby aggravating NSCLC [13,14]. Nevertheless, the defnite mechanism of M2 macrophages facilitating the NSCLC occurrence is not fully clarifed.
MicroRNAs (miRNAs) are highly conserved small noncoding RNAs [15], exert pivotal functions in various human malignancies, and are promising targets for tumor therapy [16,17]. Recently, increasing miRNAs have been confrmed to mediate the NSCLC process. For instance, miR-130a mediates macrophage polarization in NSCLC, and miR-130a loss is interrelated to NSCLC-poor prognosis and increased tumor stage [18]; miR-4319 knockdown accelerates NSCLC tumor progression by accelerating M2 macrophage polarization, which might supply promising strategies for NSCLC treatment [19].
miR-613 is a widely functional miRNA in various tumors. It has been reported that miR-613 slows down the process of cervical squamous cell cancer by targeting LETM1 [20]. miR-613 can regulate gastric cancer progression by regulating reactive oxygen species (ROS) production, glutathione content (GSH), and superoxide dismutase (SOD) activity [21]. miR-613 represses cell migration and invasion in esophageal squamous cell carcinoma via mediating G6PD to inactivate the STAT3 signaling pathway [22]. miR-613 induces NSCLC cell sensitivity to cisplatin by targeting GJA1 [23]. However, it remains unknown whether miR-613 mediates M2 macrophage polarization in NSCLC.

Cell
Transfection. miR-613 mimic and NC-mimic were obtained from Ribobio (Guangzhou, China). H1650 cells and M2 macrophages (1 × 10 6 ) were placed in six-hole plates and cultured overnight, followed by cell transfection with miR-613 mimic or NC-mimic after 24 h using Lipofectamine 2000 following the manufacturer's protocols.

Co-Culture
System. Transwell inserts with 0.4 μM aperture from Corning (New York, USA) were applied as a coculture system. THP-1 cells (2 × 10 5 ) were placed in Transwell inserts and handled with 20 ng/mL IL-4 for 2 d to diferentiate to M2 macrophages, and the miR-613 mimic was transfected into M2 macrophages. Afterward, the upper chambers were transferred into Petri dishes with H1650 cells and co-cultured for 24 h.

RNA Extraction.
Total RNA was extracted in a TRIzol reagent (Beyotime, Shanghai, China) as per standard procedure, and then, RNA content and quality were evaluated via detecting optical density (A260, A280, and A230).

Cell
Proliferation. Te proliferation of H1650 cells was determined using EdU Cell Proliferation Kits (Ribobio) following a standard procedure. H1650 cells (1 × 10 3 ) were placed in 96-well plates and cultured for one day. EdU (50 μM) was incubated with cells for two hours. Cells were then fastened with 4% paraformaldehyde (Solarbio, Beijing) and dyed by Hoechst 33342 and an Apollo reaction cocktail. Te images were obtained by fuorescence microscopy (Olympus, Tokyo, Japan). Te percentage of positive cells was counted and calculated using ImageJ2X (Rawak Software Inc.).

Flow Cytometry.
Te apoptosis condition of H1650 cells was assessed via FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, New Jersey, USA). After H1650 cells were harvested, the cells were resuspended, handled for 20 min with FITC Annexin V at 37°C, and further handled for 20 min with propidium iodide (PI) in the dark. H1650 cell apoptosis was tested with a FACSCalibur Flow Cytometer (BD Biosciences).

Wound-Healing
Assay. H1650 cells were fostered to 50% confuence in six-hole plates; the 1% double-antibody serum-free medium was replaced. Ten, the plate surface was lightly scratched by 200 μL pipette tips and put into a 37°C 5% CO 2 incubator for culture. After scratching with an inverted optical microscope (Olympus), H1650 cell migration was monitored at 0 h and 48 h. Te migration area was measured using ImageJ2X (Rawak Software Inc.), and the mobility was calculated.

Transwell Analysis.
For cell migration analysis, H1650 cells (5 × 10 4 ) were suspended in a FBS-free medium (100 μL) and transferred onto an upper chamber (Corning, Cambridge) with noncoated membranes. Lower chambers were flled with standard medium (Termo Fisher Scientifc). After incubating for 24 h, the cells on the membrane upper surface were removed and stained cells on the membrane lower surface with crystal violet (0.1%, Solarbio).
For cell invasion analysis, Matrigel chambers were conducted. After H1650 cells were harvested, they were resuspended in a serum-free medium and transferred to hydrated matrix chambers (50 μL). Te bottom chambers were put in RPMI-1640 (500 μL) containing 10% FBS. On the next day, cells on the upper surface were scraped and stained infltrating cells on the lower surface by 0.1% crystal violet (Solarbio). All cells were counted with 3-5 random felds. Ten, we use ImageJ2X (Rawak Software Inc.) to quantify.

TUNEL and Ki-67
Staining. TUNEL kit (Beyotime) was applied for TUNEL staining following instructions. Xenograft tissues were fastened in 4% paraformaldehyde, embedded in parafn, and deparafnized and rehydrated. Followed by the antigen retrieval, sections (5 μm) were incubated for 1.5 h with a 50 μL TUNEL mix at 37°C, washed, and DAPI (2 µg/mL) was added for nuclei staining, followed by washing using PBS and observing under a fuorescence microscope (OLYMPUS).
For Ki-67 staining, the abovementioned sections were handled by anti-Ki-67 (Abcam, 1 µg/mL, ab15580) at 4°C and handled by secondary antibodies (Abcam, biotin conjugated goat polyclonal vector, 1 : 250) and observed with a microscope. Mean intensities for positive Ki-67 expression were determined with Image software. Randomly selected the percentage of Ki-67 positive staining from fve felds.

Statistical
Analysis. Data are measured as mean-± standard deviation. Statistically signifcant diferences were evaluated with one-way or two-way ANOVA or an unpaired student's t-test followed by a Tukey's post-test. P < 0.05 was statistical signifcance.

miR-613 Is Lessened in NSCLC.
To clarify the miR-613 function in NSCLC, we determined the miR-613 level in NSCLC tissues and found a decreased miR-613 level in NSCLC tissues (Figure 1(a)). Meanwhile, miR-613 dropped in NSCLC cells HCC827, H1299, H1650, and A549 compared with normal human lung cell lines HLF-A, and the decrease was most notable in H1650 cells (Figure 1(b)), which shows that the diferential expression of miR-613 is most obvious in H1650 cells. Terefore, we selected H1650 cells for subsequent experiments. In summary, our study confrmed that miR-613 is lowly expressed in NSCLC tissues and cells, which suggests that miR-613 may be a potential therapeutic target for NSCLC.

miR-613 Overexpression Weakens NSCLC Cell Invasion and
Migration. Subsequently, we continued to evaluate miR-613 impact on NSCLC cell invasion and migration. As exhibited in Figure 3(a), miR-613 mimic repressed H1650 cell migration, and this conclusion was further validated by Transwell analysis (Figure 3(b)). Meanwhile, the H1650 cell invasion was restrained after the miR-613 overexpression (Figure 3(c)). To sum up, the miR-613 overexpression restrained NSCLC cell invasion and migration.
M2 macrophages usually excrete anti-infammatory factors such as TGF-β and IL-10 [30]. Tus, we further evaluated the TGF-β and IL-10 levels in the cell culture supernatant. ELISA results clarifed that the IL-10 and TGFβ contents were elevated after IL-4 treatment, while miR-613 mimics partially reversed this trend (Figure 4(b)). Te abovementioned experimental results illustrated that miR-613 restrained the M2 macrophage polarization.

miR-613 Restrains NSCLC Cell Growth by Reducing M2
Macrophage Polarization. We further determined whether miR-613 regulated the NSCLC cell growth by repressing M2 macrophage polarization. Macrophages with diferent treatments (as in Figure 4) were co-cultured with H1650 cells. Figure 5(a) presents a schematic diagram of the co-culture system. CCK-8 assay authenticated that coculture of M2 macrophages increased H1650 cell viability, while this increase was partially inverted after the miR-613 was overexpressed in M2 macrophages ( Figure 5(b)). Te same trend was discovered in the H1650 cell proliferation analysis ( Figure 5(c)). On the contrary, the co-culture of M2 macrophages weakened H1650 cell apoptosis, while the miR-613 overexpression partially reversed this efect ( Figure 5(d)). Meanwhile, the co-culture of M2 macrophages accelerated H1650 cell migration, while this trend was partially inverted after the miR-613 was overexpressed in M2 macrophages, and the Transwell assay further confrmed this fnding (Figures 5(e)-5(f )). Moreover, we further evaluated the H1650 cell invasion, and the results expounded that the co-culture of M2 macrophages facilitated H1650 cell invasion, while the miR-613 overexpression partially reversed this efect ( Figure 5(g)). In summary, miR-613 restrained NSCLC cell growth by reducing M2 macrophage polarization.   6(a) and 6(b), the co-culture of M2 macrophages facilitated tumor growth, but this trend was inverted in part after the miR-613 was overexpressed in M2 macrophages. Meanwhile, Ki67 staining confrmed that the coculture of M2 macrophages accelerated the proliferation, and the miR-613 overexpression partially reversed this trend (Figure 6(c)). On the contrary, TUNEL staining demonstrated that the co-culture of M2 macrophages restrained the apoptosis, while this restraint was partially reversed after the miR-613 was overexpressed in M2 macrophages (Figure 6(c)). Furthermore, miR-613 was decreased after co-culture of M2 macrophages, and this decrease was partially reversed by miR-613 overexpression (Figure 6(d)). Tese data confrmed miR-613 overexpression restrained tumor growth by repressing M2 macrophage polarization.

Discussion
Te high global mortality rate from NSCLC is a major issue and the challenges of cancer-related treatment [31]. Various miRNAs are pivotal regulatory molecules in NSCLC [7,32]. Similarly, our study authenticated that miR-613 was lessened in NSCLC. Furthermore, our research demonstrated that miR-613 overexpression restrained NSCLC cell proliferation, migration, invasion, and enhanced apoptosis. Meanwhile, we confrmed that miR-613 overexpression restrained NSCLC growth by repressing M2 macrophage polarization. Te completion of this research might provide promising biomarkers for NSCLC treatment. Accumulated evidence suggests that miR-613 is interrelated to various tumor genesis and development. For instance, miR-613 is lowly expressed in colorectal cancer and has diagnostic and prognostic functions in colorectal cancer [33]; miR-613 represses hepatocellular carcinoma cell dediferentiation through the SOX9 signaling, which provides novel therapeutic targets for hepatocellular carcinoma [34]. It has been authenticated that miR-613 initiates NSCLC cell cycle arrest via regulating CDK4 [35]. Similarly, we verifed    Journal of Oncology that miR-613 was lessened in NSCLC cells and tissues. Simultaneously, we further confrmed that the transfection of miR-613 mimic advanced cell apoptosis and weakened NSCLC cell proliferation, migration, and invasion through various gain-of-function assays, which was similar to a previous conclusion [36]. Previous reports state that macrophages with carcinogenic functions in tumor environments are considered as TAMs and typically exhibit an M2 phenotype [37,38]. Increasing studies authenticate that M2 macrophages are interrelated to multiple tumors development. For instance, Sousa et al. confrmed that the high number of M2 macrophages is relevant to rapid proliferation and poor prognosis of human primary breast tumors [39]; Yamaguchi et al. clarifed that the peritoneal TAMs polarization into M2 phenotype facilitates the gastric cancer tumor growth, prompting that intraperitoneal TAMs is a promising target for gastric cancer [40]. Recently, the pivotal function of M2 macrophage polarization in NSCLC has attracted widespread attention from researchers. M2 macrophages activate the ERK1/2/Fra-1/slug pathway through epithelialmesenchymal transformation to accelerate the malignant development of NSCLC [41]. Puerarin reduces the M2 macrophage metastasis and polarization of NSCLC transplant-tumor-associated macrophages by inactivating MEK/ ERK 1/2 signaling, thereby alleviating NSCLC [42]. Considering the critical functions of miR-613 and M2 macrophages in NSCLC, this study attempted to clarify whether miR-613 alleviated NSCLC by mediating M2 macrophage polarization. As expected, our experimental data revealed that miR-613 restrained NSCLC growth through repressing M2 macrophage polarization.  Figure 6: miR-613 infuences tumor growth by reducing the M2 macrophage polarization in vivo. Macrophages with diferent treatments (as in Figure 4) were co-cultured with H1650 cells. H1650 cells were grouped: H1650+M NCmimic , H1650+M IL-4+ NCmimic and H1650+M IL-4+miR-613mimic . Te C57BL/6 mice were subcutaneously injected with the above H1650 cells ( However, there are still some limitations in this study, and although, we found that miR-613 regulated NSCLC development by inhibiting M2 macrophage polarization, whether miR-613 would regulate NSCLC development by regulating downstream gene targets or oxidative stress response to inhibit M2 macrophage polarization still needs further investigation.
In general, we confrmed miR-613 overexpression enhanced apoptosis and restrained NSCLC cell proliferation, migration, and invasion. Meanwhile, miR-613 overexpression restrained NSCLC growth by repressing M2 macrophage polarization, which provides novel insights and strategies for treating NSCLC.

Data Availability
Te data used to support the fndings of this study of the study are available from the corresponding author upon reasonable request.

Ethical Approval
Te experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration. All programs about animal are approved by the Animal Ethics Committee of Afliated Hospital of Nantong University.

Consent
All patients signed an informed consent form before performing surgery and the study was approved by the Ethics Committee of Afliated Hospital of Nantong University.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
MJY and WZ conceived and designed the study. MMX, XH, and YYS conducted most of the experiments. MS and ZPW analyzed the data. MJY performed the literature search and data extraction. MJY drafted the manuscript. MJY and WZ fnalized the manuscript. All authors read and approved the fnal manuscript. Mingjun Yang and Wen Zhou contributed equally to this work.